Friedel Roland H
Helmholtz Center Munich, Institute of Developmental Genetics, Neuherberg, Germany.
Methods Mol Biol. 2009;561:185-97. doi: 10.1007/978-1-60327-019-9_12.
Gene targeting of mouse embryonic stem cells facilitates the deliberate engineering of the mouse genome. The gene targeting vector is introduced into cells by electroporation, and cell clones carrying the targeting vector are obtained by drug selection. Clones can be screened effectively for correct recombination events by either Southern blot analysis with external and internal probes, or by long-range PCR. Positive clones are subsequently expanded and prepared for the generation of chimeric mice.
小鼠胚胎干细胞的基因打靶有助于对小鼠基因组进行精心改造。通过电穿孔将基因打靶载体导入细胞,并通过药物筛选获得携带打靶载体的细胞克隆。可通过使用外部和内部探针的Southern印迹分析或通过长距离PCR有效地筛选克隆中的正确重组事件。随后对阳性克隆进行扩增,并为生成嵌合小鼠做准备。